Screening methods

ABSTRACT

The present invention provides materials and methods relating to screening for compounds useful in the treatment of Alzheimer&#39;s disease and related conditions. In particular, screening methods using tyrosine kinases are provided, as are methods relating to the role of tyrosine kinases as therapeutic targets.

FIELD OF THE INVENTION

The present invention relates to screening methods, and moreparticularly to methods which relate to the role of tyrosine kinases astherapeutic targets for Alzheimer's disease and related conditions.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a neurodegenerative disease characterised bythe presence of senile plaques and neurofibrillary tangles in the brain.The degree of dementia at death correlates better with neurofibrillarytangle numbers and with neuronal and synaptic loss than with senileplaque counts. The presence of neurofibrillary tangles in neuronsresults in the death of those neurons, implying that prevention oftangle formation is an important therapeutic goal. The principal proteinthat forms the neurofibrillary tangle is the microtubule-associatedprotein, tau, which assembles into filaments that have the appearance oftwisting about each other in pairs and are referred to as paired helicalfilaments (PHF). PHF are present in different locations in degeneratingneurons in the Alzheimer brain and when many aggregate in the neuronalcell body, they produce the neurofibrillary tangle (Lee et al., 2001).

Senile plaques have an extracellular central deposit of amyloidβ-peptide (Aβ), which is surrounded by dystrophic neurites to form thesenile or neuritic plaque. In vitro and in vivo Aβ has been shown to beneurotoxic. Aβ is derived by proteolytic processing of the largeramyloid precursor protein (APP). Much attention has been focused on Aβproduction as a therapeutic target because its production is believed tobe an early event in AD pathogenesis. This is because mutations in theAPP gene, which give rise to autosomal dominant AD, result in eitherincreased overall production of Aβ or in a relative increase in theslightly longer Aβ₄₂ over Aβ₄₀, the former being more amyloidogenic;Aβ₄₂ has two additional hydrophobic amino acids at the C-terminus of40-residue Aβ₄₀ thereby endowing the peptide with an increased tendencyto aggregate and form amyloid fibres. Mutations in two other genes thatalso cause autosomal dominant AD, presenilin-1 and presenilin-2 (PS1 &PS2) also result in an increase in the ratio of Aβ₄₂ to Aβ₄₀. The beliefthat Aβ deposition in the brain precedes the appearance ofneurofibrillary tangles has been the basis of the amyloid cascadehypothesis but it has been uncertain whether tangles are important inpathogenesis or are only an unimportant epiphenomenon. This has beenchanged by the discovery of mutations in the gene for tau in some otherrelated neurodegenerative diseases.

The mechanism by which Aβ kills neurons in the brain has still to beestablished. Many studies of Aβ toxicity have been conducted in tissueculture using rat brain neuronal cultures. We have shown that exposureof both foetal rat and human brain neuronal cultures to aggregated Aβinduces within 2 to 10 minutes increases in the phosphotyrosine contentof several proteins including tau (Williamson et al., 2002). We havealso shown that this treatment results in activation of the tyrosinekinases FAK and Fyn, the latter being a member of the src family oftyrosine kinases. This tyrosine phosphorylation of tau was prevented byinhibitors that act on the src family of tyrosine kinases and act onc-Abl.

It has previously been reported that increased levels of Fyn areassociated with neurons containing abnormally phosphorylated tau in ADbrain (Shirazi and Wood, 1993) and we have demonstrated using antibodiesthat recognise phosphotyrosine that PHF-tau from AD brain containsphosphotyrosine (Williamson et al., 2002). There are five potentialsites for tyrosine phosphorylation in tau, these are residues 18, 29,197, 310 and 394, based upon the numbering of residues in the longesthuman brain isoforms of tau of 441 amino acids. We have shown in vitrothat Fyn and Lck, both src family kinases, phosphorylate recombinanthuman tau and phosphotyrosines 18, 197, 310 and 394 were positivelyidentified in one or more of their respective tryptic peptides, fromsequence information of fragmented peptides (Scales et al., 2002).

Neurons in brain slices from transgenic mice in which the Fyn gene hasbeen disrupted are resistant to Aβ toxicity (Lambert et al., 1998).Thus, there is evidence that activation of Fyn may be involved in Aβtoxicity.

It has been reported that Aβ treatment of microglia in culture resultsin activation of several other tyrosine kinases, namely Syk, Lyn and FAK(McDonald et al., 1997) and, as mentioned above, we have found that FAKis also activated in primary neurons exposed to Aβ (Williamson et al.,2002). Syk has been reported to phosphorylate α-synuclein on tyrosine,α-synuclein being the principal protein of Lewy bodies which are thepathological hallmark of Parkinson's disease and are also present in upto 70% of AD brains (Negro et al., 2002). Finally, we have found thatthe protein tyrosine kinase Abl phosphorylates tau in co-transfectedcells and Abl is implicated in activation of the serine/threonineprotein kinase cdk5, which is regarded as a pathogenically important taukinase that phosphorylates many residues in tau that can alternativelybe phosphorylated by GSK-3 (Zukerberg et al., 2000). Thus, there is thestrong possibility that tau is a substrate for various tyrosine kinasesand that these need to be considered in the context of the possiblepathogenesis of the tauopathies.

The presence of intraneuronal deposits of tau in the form of typicalneurofibrillary tangles in AD or other morphologically distinct tauaggregates in a number of other neurodegenerative diseases, is the basisfor grouping these conditions as tauopathies. Thus, in addition to AD,the main examples of the tauopathies are frontotemporal dementia withParkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclearpalsy (PSP), Pick's disease, corticobasal degeneration, and multisystematrophy (MSA). The intracellular tau deposits (usually neuronal butsometimes also glial) are filamentous and in a hyperphosphorylated statecompared to the phosphorylation of tau in control human brain. In thecase of AD, this hyperphosphorylated tau is often referred to as PHF-taubecause it is derived from the PHF.

Other than for AD, deposits of Aβ in the brain are either absent orminimal in these other tauopathies. There are some tauopathy pedigreeswith autosomal dominant disease in which the causative gene has beenidentified as the tau gene and although cases with the same mutation maypresent with apparently different diseases, they invariably have taudeposits in the brain and are mostly of the FTDP-17 variety. Thus, thefinding of mutations in the tau gene which result in disease anddeposition of tau aggregates in neurons is compelling evidence for theprimary pathogenic importance of tau deposition in all of theseconditions, including AD, whatever the primary cause of disease.Therefore, the amyloid cascade hypothesis is borne out by the discoveryof tau mutations and confirms that indeed neurofibrillary tangleformation may well be subservient to Aβ deposition in AD, but that inthe other tauopathies lacking Aβ deposits, then some other primary eventmust trigger the tau pathology. Tau abnormalities and deposition aretherefore important therapeutic targets for all tauopathies, includingAD.

Tau is a phosphoprotein, the function of its phosphorylation remainingto be unequivocally established. However, increased phosphorylation oftau on multiple serine and threonine residues reduces the ability of tauto promote microtubule assembly and to stabilise assembled microtubules,effects that have been demonstrated both in vitro and in cells. Manystudies have shown that PHF-tau from AD brain is more heavilyphosphorylated on serine and threonine than tau from control brain. Thishas been demonstrated partly by protein sequencing and partly bydemonstrating that certain monoclonal antibodies only label eitherPHF-tau or alternatively they label non-phosphorylated tau and notPHF-tau; the epitopes for many of these antibodies have been mapped toparticular phosphorylated residues present in PHF-tau and absent from,or present at lower levels in, control brain tau. The pathological taufrom most other cases of other tauopathies seems to be similarlyhyperphosphorylated to PHF-tau.

These findings strongly imply that similar abnormalities in regulatingphosphorylation of tau are shared by all the tauopathies including AD.Since phosphorylation of proteins is effected by protein kinases anddephosphorylation by protein phosphatases, identifying the proteinkinases and phosphatases for tau is important because these enzymes arepotential therapeutic targets for these diseases.

As mentioned above, there are five tyrosines in human brain tau. It hasbeen reported that Fyn phosphorylates tau in non-neuronal co-transfectedcells and that tyrosine 18 is the preferred phosphorylation site (Lee etal., 1998). We have reported that PHF-tau isolated from Alzheimer brainis phosphorylated on tyrosines and others have identified tyrosine 18 asone site of phosphorylation (Williamson et al., 2002; Lee et al., 2004).

Cultured neurons from transgenic mice in which the tau gene has beendisrupted, such that these animals no longer express the tau protein,are resistant to exposure to Aβ and do not die (Rapoport et al., 2002).This requirement of tau for Aβ to be neurotoxic has been confirmed inexperiments in which neurons treated with antisense oligonucleotides toreduce expression of tau were resistant to the neurotoxic effects of Aβexposure (Liu et al., 2004).

It remains a considerable problem in the art in identifying the enzymesresponsible for causing phosphorylation of paired helical filament tauand the sites phosphorylated by those enzymes.

SUMMARY OF THE INVENTION

Broadly, the present invention relates to the modulation of thephosphorylation of tau protein at tyrosine sites through its interactionwith kinases. In particular, the present invention concerns theidentification of tyrosine phosphorylation sites in tau protein and thekinases that preferentially phosphorylate subsets of those sites (e.g.tyrosine kinases). This identifies novel therapeutic targets andinteractions that can be employed in methods of screening for candidatetherapeutic agents. In one aspect, the present invention is based on theidentification of the role played by the protein tyrosine kinase c-Ablin the phosphorylation of tyrosine 394 in PHF tau. This has notpreviously been disclosed in the prior art. Prior to the presentinvention, the prior art proposed that tyrosine 394 was phosphorylatedby Fyn, another Src family protein tyrosine kinase. Thus, in contrast toprior art approaches based on screening for compounds capable ofinhibiting tyrosine 394 phosphorylation by inhibition of Fyn kinase, inone aspect, the present invention provides methods of screening forsubstances useful in the treatment of AD, or another tauopathy, whichare inhibitors of c-Abl.

Some of the work described herein involved the technically difficultdetermination of tau phosphorylation state in PHF tau from Alzheimer'spatients brains, rather than more conventional approaches employingnormal tau or fetal brain tau. Accordingly, the present inventionprovides the first disclosure of the presence of tyrosinephosphorylation in clinical PHF tau, and in some aspects, the first tolink a specific kinase with a specific phosphorylation event.

Furthermore, the identification herein of c-Abl as a candidate for atarget to treat AD is based on modifying the disease associated tyrosinephosphorylation of tau and is further supported by the observation thatc-Abl may be a source of cdk5 activation since cdk5 is proposed as analternative to GSK-3 in the production of PHF tau.

Furthermore, the present invention proposes that fyn, lck and c-Abl arecandidate targets for drugs to treat AD or other tauopathies because ofthe work described herein which links these kinases to the amyloidcascade theory through their recruitment to lipid rafts. Without wishingto be bound by any particular explanation, the present inventors proposethat Aβ activates these tyrosine kinases through interaction withcholesterol-rich domains of cell membranes and that this results in aninappropriate over-association of tau with these regions of membranesand a subsequent disturbance of intracellular cell signalling processes.The kinases involved in these signalling events can therefore be usedalone or in any combination as therapeutic targets for the screening ofmodulators of the activity and/or their interaction with tau.

Accordingly, the present invention provides methods of screening forcandidate compounds useful in the treatment of Alzheimer's disease or atauopathy that act by inhibiting specific phosphorylation of tauprotein. These methods can be carried out in many ways includingmeasurement by mass spectrometry and immunoassay.

Considering the requirements described above for the obligatoryexpression of both Fyn and tau in order for Aβ to be neurotoxic and thepreviously known fact that Fyn can phosphorylate tau in cells, thepresent inventors propose that a sequence of biochemical events isinvolved in the processing of tau. This is that exposure of neurons toAβ induces activation of Fyn and probably other protein tyrosinekinases, which then phosphorylates tau and this results in a series offurther biochemical changes ending in neuronal cell death, which mayinvolve hyperphosphorylation of tau on numerous serine and threonineresidues.

The first stage in identifying the responsible enzymes is to map all ofthe phosphorylation sites in PHF-tau and compare the complement of siteswith those in control brain tau. Protein sequencing studies have intotal resulted in the identification of 25 phosphorylation sites inPHF-tau (Hanger et al., 1998; Morishima-Kawashima et al., 1995); controlbrain tau has not been studied as extensively and only a few of thesesites have been identified in tau from adult control human brain or fromfoetal control human brain (tau from foetal brain is known to be morephosphorylated than that from adult brain).

As described above, the prior art disclosed that of the five potentialtyrosine phosphorylation sites present in human tau protein at positions18, 29, 197, 310 and 394, the tyrosine kinases Lck and Fyn phosphorylatetau at tyrosine positions 18, 310 and 394 and that tyrosine 18 is thepreferred phosphorylation site of Fyn. However, the present inventiondiscloses for the first time that PHF-tau from AD brain isphosphorylated at Tyr-394, a new result arising from mass spectrometryexperiments. The present invention also demonstrates that Fynphosphorylates primarily Tyr-18 in cells, and that Abl phosphorylatesprimarily Tyr-394. These findings mean that the phosphorylation ofTyr-394 may contribute to AD pathology and that Abl is a potential drugtarget.

Accordingly, the work described in the present application refines theinitial indications provided in the prior art and to investigate how Aβmay trigger activation of Fyn, how Fyn might come into contact with tau,and on which particular tyrosine residues in tau Fyn, and the kinasesSyk and Abl, might act.

Aβ Neurotoxicity—Lipid Rafts

Fyn is known to be associated with lipid rafts, which are domains ofcell membranes rich in cholesterol and sphingolipids. Solubilising cellsin certain detergents such as Triton X100 at 4° C. enables isolation oflipid rafts since these cholesterol-rich domains remain insoluble andcan be separated from other cell components by virtue of their lowbuoyant density. Thus, lipid rafts are isolated by flotation on sucrosesolutions by ultracentrifugation. Furthermore, it has been reported thatbinding of Aβ to membranes is mediated, at least in part, by cholesteroland that increasing membrane cholesterol levels is positively correlatedwith Aβ toxicity to neuronal and endothelial cells (Eckert et al., 2000;Subasinghe et al., 2003; Wang et al., 2001; Yip et al., 2001).Flotillin, a lipid raft constituent, accumulates in tangle-bearingneurons in Alzheimer brain indicating abnormalities in lipid rafts inthe diseased brain (Girardot et al., 2003), and indeed the proteincomposition of lipid rafts isolated from Alzheimer brain has beenreported to be abnormal (Ledesma et al., 2003). We have investigatedlipid rafts in the context of Aβ neurotoxicity.

The present application describes investigations into the effects onlipid rafts of exposing neurons to Aβ. To summarise, we have found bywestern blotting that lipid rafts isolated from primary cultures of ratbrain cortical neurons contain the marker protein, flotillin, as well asFyn, FAK, and small but reproducible amounts of actin, tubulin and tau.After exposure to 10 μM Aβ for 5 min, there is an increase in thephosphotyrosine content of numerous proteins, as detected with thephosphotyrosine monoclonal antibody 4G10; there are also increases inthe amounts of Fyn, FAK, tau, tubulin, actin and c-Src kinase, but notβ-catenin, relative to the flotillin content of lipid rafts compared tountreated neurons.

We have also found that pre-treatment of neuronal cultures with the Srcfamily tyrosine kinase inhibitor, PP2, before exposure to Aβ andsubsequent isolation of lipid rafts, resulted it blocking of therecruitment of increased quantities of tau and Fyn to the lipid raftsthat was induced by Aβ.

Tyrosine Phosphorylation of Tau

Fyn has previously been shown to phosphorylate tau in cellsco-transfected with tau and Fyn (Lee et al., 1998). As mentioned above,we previously found that in vitro Fyn and Lck phosphorylate human tau onfour of the five tyrosines present in human tau (Y18, Y197, Y310, Y394)(Scales et al., 2002). We have now made a series of mutant forms of tauin which either each of the five tyrosines was individually mutated tophenylalanine (F18, F29, F197, F310, F394) or in which only a singletyrosine remained with the other four replaced by phenylalanine(Y18-only, Y29-only, Y197-only, Y310-only, Y394-only).

Using these mutants, we have found that by treating non-neuronal cellstransfected with these mutant forms of tau with pervanadate to inhibittyrosine phosphatases, there is an increase in endogenous tyrosinephosphorylation of tau, principally on tyrosine 394 with a contributionfrom tyrosine 197. In other experiments in which mutant forms of tauwere co-transfected with Fyn, Syk or Abl tyrosine kinases, we foundpreferential phosphorylation of tyrosine 18 and 310 by Fyn, tyrosines18, 29, 197 and 394 by Syk but Abl phosphorylated preferentiallytyrosines 197, 310 and 394.

Using rat brain lysate in the presence of pervanadate to phosphorylaterecombinant human tau in vitro, we have found by mass spectrometry thattyrosines 310 and 394 were phosphorylated.

Finally, tyrosine phosphorylation in tau is a physiological event sincewe have found by mass spectrometry unequivocal evidence that tyrosine394 is phosphorylated in tau isolated from human foetal brain and inPHF.

Tyrosine Phosphorylation of Tau Generates an SH2 Binding Site for Fyn

We have found that in vitro phosphorylation of tau by Lck generates abinding site for the SH2 domain of Fyn. In summary, the evidencesuggests that more than one tyrosine kinase phosphorylates tau, withdifferent kinases preferentially phosphorylating different tyrosineresidues, and that Aβ is capable of activating at least some of thosekinases. The data also demonstrate that tyrosine phosphorylation of taugenerates a binding site for at least one tyrosine kinase, implying thattau may be an important cell signalling protein in addition to its roleas a microtubule-associated protein.

Accordingly, in one aspect, the present invention proposes that threekinases phosphorylate tau protein at the tyrosine phosphorylation sitesat Tyr18, Tyr29, Tyr197, Tyr310 and Tyr394. The kinases are Fyn, Syk andAbl. A description and the sequences of these kinases are provided in:

Fyn: Semba, K. et al (1986) Proc. Natl. Acad. Sci. USA 83, 5459-5463.See Genbank NM_(—)002037 and that there are two main isoforms. Thepresent invention is primarily concerned with the isoform expressed inbrain, but the other isoform expressed in haematopoietic cells, such asT cells, may also find use in the method of screening disclosed herein.

Syk: Law, C. L. et al, J. Biol. Chem. 269, 12310-12319. See GenbankL28824.

c-Abl: Fainstein, E., Einat, M., Gokkel, E., Marcelle, C., Croce, C. M.,Gale, R. P. and Canaani, E. (1989) Oncogene 4, 1477-1481. See GenbankX16416 and M14752. There are several isoforms involving the N-terminus,but having a similar catalytic domain.

In referring to these kinases, the present invention includes the use ofisoforms, splice variants, fragments and sequence variants, as discussedin more detail below.

In particular, the kinases and the sites they preferentiallyphosphorylate can be used in methods of screening for inhibitors ofphosphorylation or promoters of dephosphorylation. Preferably, thescreening method is for finding substances which are capable ofinhibiting phosphorylation. The screening method may involve determiningwhether a candidate substance is capable of binding to the kinase and/ortau protein, e.g. to inhibit or prevent the phosphorylation of tauprotein at a given site by the kinase in question. This method mayinvolve contacting the candidate substance with the kinase and/or tauprotein and determining whether binding occurs, and optionally theaffinity of the binding reaction. Alternatively or additionally, themethod may comprise determining whether a candidate substance is capableof inhibiting a kinase, e.g. to inhibit or prevent the phosphorylationof a substrate such as a tau protein at a site by one of the kinases, asdisclosed herein. This determination may comprise contacting a candidatesubstance with the kinase in question and tau protein or an alternativesubstrate (e.g. a fragment of tau comprising the amino acid sequencearound the phosphorylation site), and determining whether the candidatesubstance inhibits the kinase phosphorylating the substrate. Thedetermining step may comprise determining the extent of the inhibition.In situations where an initial screen is carried out to identifycandidate substances which are capable of binding to tau protein or akinase, or are capable of inhibiting the activity of a kinase, themethod may comprise the further step of determining whether the bindingor inhibiting property of the candidate substance is capable ofinhibiting the phosphorylation of tau protein or a fragment thereof inthe presence of the kinase.

The screening for candidate substances having these properties mayemploy tau protein, or a fragment, active portion or sequence variantthereof comprising one or more of the relevant phosphorylation sites.One example of a tau protein that may be employed in this way is afragment of tau comprising the amino acid sequence around thephosphorylation site.

The sites and the kinases that preferentially phosphorylate them aretyrosines 18 and 310 by Fyn, tyrosines 18, 29, 197 and 394 by Syk, andtyrosines 197, 310 and 394 by Abl.

As a consequence of these findings, the new sites and kinases can beused as the basis of assays and assays methods for screening formodulators of the phosphorylation of the sites in tau protein for use ordevelopment as therapeutics for the treatment of tauopathies. As a firststep, the candidate substances may be tested to determine whether theyare inhibitors or promoters of the kinases disclosed herein. Optionally,the method may alternatively or additionally comprise determiningwhether a candidate substance is capable of inhibiting thephosphorylation of tau by a kinase and/or promoting thedephosphorylation of phosphorylated tau by a phosphatase (e.g. atyrosine phosphatase).

Accordingly, in a further aspect, the present invention provides the useof (a) a kinase which is capable of phosphorylating tau protein at theone or more of the sites disclosed herein and (b) a substrate of thekinase, wherein the kinase and substrate are used for identifyingcandidate substances which are capable of inhibiting phosphorylation ofthe substrate by a kinase.

In the present invention, the tau protein comprising the phosphorylationsites may be substantially full length and/or wild, type tau or PHF tauprotein, or may be a fragment, active portion or sequence variantthereof. In other embodiments, the present invention may employ acorresponding nucleic acid molecule encoding the tau protein. Where atau protein which is a fragment, active portion or sequence variant isemployed, the phosphorylation site(s) may be present with surroundingamino acids from the tau protein sequence. Preferably, the presentinvention employs PHF tau protein. In the present invention thenumbering of tau and PHF tau is according to the sequence disclosedFigure 1 of Goedert et al (1989) Neuron 3, 519-526: Multiple isoforms ofhuman microtubule-associated protein Tau: sequences and localisation inneurofibrillary tangles of Alzheimer's Disease Goedert M, Spillantini MG, Rutherford D, Jakes R and Crowther R A.

Alternatively or additionally, any of the above defined tau proteins maypossess phosphorylation at one or more of the phosphorylation sites.This enables the effects of cooperative phosphorylation of the proteinto be studied, that is, where the phosphorylation of one site isdependent in changes to the tau protein caused by one or more precedingor simultaneous phosphorylation steps. Thus, in some embodiments of thepresent invention, the tau protein may include one or more of the knowntau phosphorylation sites.

In a further aspect, the present invention provides a method ofscreening for substances which are capable of inhibiting phosphorylationat one or more of the site(s) of a substrate by a kinase, the methodcomprising:

(a) contacting at least one candidate substance, a kinase which iscapable of phosphorylating tau protein at the one or more of the sitesdisclosed herein and a substrate of the kinase;

(b) determining whether, and optionally the extent to which, thecandidate substance inhibits the phosphorylation of the substrate by thekinase; and,

(c) selecting the candidate substance which inhibits phosphorylation ofthe substrate.

The method disclosed herein may be employed for identifying candidatesubstances useful in treating or developing lead compounds for treatingtauopathies.

In all aspects of the invention, the substrate may be a tau protein, orcomprise a fragment of tau protein, which includes one or more of thephosphorylation site(s) acted on by the kinase. For example, in the caseof c-Abl, the substrate may be a fragment of tau protein based on theamino acid sequence surrounding Tyr 394. However, in other embodiments,other non-tau based substrates of the kinase may be employed, forexample where a substrate of the kinase is readily available. In thiscase, the method may comprise the further step of confirming whether acandidate substance selected in an initial screen has the property ofinhibiting the phosphorylation of the tau protein under conditions inwhich the kinase is capable of phosphorylating the site(s) of the tauprotein in the absence of the candidate substance.

In this embodiment, the method may additionally involve including aphosphatase inhibitor in step (a) to inhibit phosphatases that may bepresent in the system from dephosphorylating the tau protein.

In a further aspect, the present invention provides a method ofscreening for substances which are capable of promotingdephosphorylation at one or more of the site(s) of a substrate by aphosphatase, the method comprising:

(a) contacting at least one candidate substance, a phosphatase which iscapable of dephosphorylating tau protein at the one or more of the sitesdisclosed herein and a substrate of the phosphatase;

(b) determining whether, and optionally the extent to which, thecandidate substance promotes the dephosphorylation of the substrate bythe phosphatase; and,

(c) selecting the candidate substance which promotes thedephosphorylation of the substrate.

In this embodiment, the method may additionally involve including akinase inhibitor in step (a) to inhibit kinases that may be present inthe system from phosphorylating the tau protein.

Examples of screening techniques suitable for use according to thepresent invention will be well known to the skilled person. By way ofexample, a cell based screening assay may be carried out byco-transfecting cells with nucleic acid encoding tau and encoding one ormore of Fyn, Syk or Abl tyrosine kinases, and determining the effectthat candidate compounds have on tau phosphorylation, in particular attyrosines 18 and 310 by Fyn, tyrosines 18, 29, 197 and 394 by Syk andtyrosines 197, 310 and 394 by Abl. Preferred methods of screening mayinvolve the use of mass spectroscopy to determine the phosphorylation atsites of tau, and this is described in detail below. Conveniently, themethods of screening may be carried out in a multiplex assay format inwhich a solid phase is employed on which a plurality of substrates areimmobilised (e.g. in an array), the substrates corresponding tophosphorylation sites of tau. By way of example, the substrates maycomprise fragments of tau protein. This is described in more detailbelow. The present invention therefore provides a kit or solid phaseadapted for carrying out a multiplex screening assay according to thepresent invention.

In some embodiments, the method may comprise, having identified acandidate substance according to one of the methods disclosed herein,the further step(s) of optimising the candidate substance to improve oneor more of its properties and/or formulating it as a pharmaceutical.

The methods and uses disclosed herein employ one of more kinasesselected from Fyn, Syk or Abl. However, the screening method maycomprise investigating the effect of one or more further enzymes onphosphorylation sites of tau. Examples of suitable further enzymes foruse in any aspect of the invention are provided below in the section onmultiplex assays.

In a further aspect, the invention provides for the use of a modulatorof tau protein phosphorylation obtainable by the methods describedherein in the treatment of a tauopathy. Preferably, the modulator is aninhibitor of tau protein phosphorylation.

In a related aspect, the invention provides for the use of a c-Abl, Sykor Fyn inhibitor in the preparation of a medicament for the treatment ofa tauopathy.

In the present invention, preferably the step of detecting the presenceand extent of phosphorylation and dephosphorylation in the tau proteincan be carried out using mass spectroscopy as described in detail below.Alternatively, or additionally, site specific recognition agents whichare capable of distinguishing between a site which is phosphorylated andone which is not may be used. Examples of such agents known in the artare site specific antibodies such as monoclonal antibody AT100.

In a further aspect, the present invention provides a substanceobtainable from one of the methods disclosed herein which is capable ofinhibiting the phosphorylation or promoting the dephosphorylation of atau protein at one or more of the above defined sites.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid sequence of the human tau isoform used forthe numbering given in this application. The phosphorylation sites Y18,Y29, Y197, Y310 and Y394 are indicated in bold.

FIG. 2 shows the amino acid sequence of the p150 isoform of human c-Abl.

FIG. 3 shows the amino acid sequence of human Syk.

FIG. 4 shows the amino acid sequence of isoform 1 of human Fyn.

Embodiments of the present invention will now be discussed in moredetail by way of example and not limitation.

DETAILED DESCRIPTION Tau Proteins

The assays and assay methods disclosed herein can employ wild-type orfull length tau proteins, kinases or phosphatases or fragments, activeportions or derivatives thereof. In the case of tau proteins, thematerials used in the assays may be unphosphorylated or partiallyphosphorylated as discussed above.

In the present invention, derivatives of the tau proteins, kinases(especially Fyn, Syk and Abl) or phosphatases have an amino acidsequence which differs by one or more amino acid residues from thewild-type amino acid sequence, by one or more of addition, insertion,deletion and substitution of one or more amino acids. Thus, variants,derivatives, alleles, mutants and homologues, e.g. from other organisms,are included. Thus, a derivative of tau protein or kinase may include 1,2, 3, 4, 5, greater than 5, or greater than 10 amino acid alterationssuch as substitutions with respect to the wild-type sequence.

Preferably, a fragment or derivative of a protein used in the assaysdisclosed herein shares sequence identity with the corresponding portionof the relevant wild-type sequence of the protein, and preferably has atleast about 60%, or 70%, or 75%, or 80%, or 85%, 90% or 95% sequenceidentity. Preferred fragments comprise at least 5, at least 10, at least15, at least 20 or at least 25 amino acids which correspond to or sharesequence identity with tau protein. Optionally, the fragments maycomprise a fragment of tau protein linked or conjugated to othermoieties, for example expression tags, purification tags, groups toenable the fragment to be immobilised or otherwise manipulated, orlabels. As is well-understood, identity at the amino acid level isgenerally in terms of amino acid identity which may be defined anddetermined by the TBLASTN program, of Altschul et al. (1990) J. Mol.Biol. 215: 403-10, which is in standard use in the art.

Identity may be over the full-length of the relevant peptide or over acontiguous sequence of about 5, 10, 15, 20, 25, 30, 35, 50, 75, 100 ormore amino acids, compared with the relevant wild-type amino acidsequence. Alternatively, nucleic acid encoding a fragment or derivativemay hybridise to the corresponding wild type nucleic acid understringent conditions, for example as disclosed in textbooks such asAusubel, Short Protocols in Molecular Biology, 1992 or Sambrook et al,Molecular Cloning, A Laboratory Manual, Cold Spring Harbour LaboratoryPress, 1989, using a hybridization solution comprising: 5×SSC,5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmentedsalmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide.Hybridization is carried out at 37-42° C. for at least six hours.Following hybridization, filters are washed as follows: (1) 5 minutes atroom temperature in 2×SSC and 1% SDS; (2) 15 minutes at room temperaturein 2×SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37° C. in 1×SSC and 1%SDS; (4) 2 hours at 42-65° C. in 1×SSC and 1% SDS, changing the solutionevery 30 minutes.

One common formula for calculating the stringency conditions required toachieve hybridization between nucleic acid molecules of a specifiedsequence homology is (Sambrook et al., 1989):

T _(m)=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63 (% formamide)−600/#bp induplex.

As an illustration of the above formula, using [Na+]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the T_(m) is 57° C. The T_(m) of a DNA duplex decreases by 1-1.5°C. with every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42° C. Such a sequence would be considered substantiallyhomologous to the nucleic acid sequence of the present invention.

Methods of Screening for Inhibitors and Enhancers

It is well known that pharmaceutical research leading to theidentification of a new drug may involve the screening of very largenumbers of candidate substances, both before and even after a leadcompound has been found. This is one factor which makes pharmaceuticalresearch very expensive and time-consuming. Means for assisting in thescreening process can have considerable commercial importance andutility.

As detailed above, methods of screening for a substance which areinhibitors of phosphorylation of tau protein or promoters ofdephosphorylation of tau protein can be carried out by contacting one ormore test substances with the tau protein and kinase or phosphatase (asdefined herein) in a suitable reaction medium, and determining thepresence or extent of phosphorylation of dephosphorylation in thepresence and absence of the candidate substance. A difference inactivity in the presence and absence of the candidate substance isindicative of a modulating effect.

Preliminary assays in vitro may be followed by, or run in parallel with,in vivo assays.

Of course, the person skilled in the art will design any appropriatecontrol experiments with which to compare results obtained in testassays.

Performance of an assay method according to the present invention may befollowed by isolation and/or manufacture and/or use of a compound,substance or molecule which tests positive for ability to modulateinteraction between one of the phosphorylation sites of tau protein (asdefined herein) and a kinase (as disclosed herein) or a phosphatase.

The precise format of an assay of the invention may be varied by thoseof skill in the art using routine skill and knowledge. For example,interaction between substances may be studied in vitro by labelling onewith a detectable label and bringing it into contact with the otherwhich has been immobilised on a solid support. Suitable detectablelabels, especially for peptidyl substances include ³⁵S-methionine whichmay be incorporated into recombinantly produced peptides andpolypeptides. Recombinantly produced peptides and polypeptides may alsobe expressed as a fusion protein containing an epitope which can belabelled with an antibody.

The protein which is immobilized on a solid support may be immobilizedusing an antibody against that protein bound to a solid support or viaother technologies which are known per se. A preferred in vitrointeraction may utilise a fusion protein includingglutathione-S-transferase (GST). This may be immobilized on glutathioneagarose beads. In an in vitro assay format of the type described above atest compound can be assayed by determining its ability to diminish theamount of labelled peptide or polypeptide which binds to the immobilizedGST-fusion polypeptide. This may be determined by fractionating theglutathione-agarose beads by SDS-polyacrylamide gel electrophoresis.Alternatively, the beads may be rinsed to remove unbound protein and theamount of protein which has bound can be determined by counting theamount of label present in, for example, a suitable scintillationcounter.

The amount of a candidate substance which may be added to an assay ofthe invention will normally be determined by trial and error dependingupon the type of compound used. Typically, from about 0.001 nM to 1 mMor more concentrations of putative inhibitor compound may be used, forexample from 0.01 nM to 100 μM, e.g. 0.1 to 50 μM, such as about 10 μM.Greater concentrations may be used when a peptide is the test substance.Even a molecule which has a weak effect may be a useful lead compoundfor further investigation and development.

Combinatorial library technology provides an efficient way of testing apotentially vast number of different substances for ability to modulateactivity of a polypeptide. Such libraries and their use are known in theart. Compounds which may be used may be natural or synthetic chemicalcompounds used in drug screening programmes. Extracts of plants whichcontain several characterised or uncharacterised components may also beused.

Antibodies directed to the site of interaction in either protein form afurther class of putative inhibitor compounds. Candidate inhibitorantibodies may be characterised and their binding regions determined toprovide single chain antibodies and fragments thereof which areresponsible for disrupting the interaction. Antibodies may also beemployed as site specific recognition agents for determining whetherphosphorylation of a site in tau protein has occurred during as assay.

Antibodies may be obtained using techniques which are standard in theart. Methods of producing antibodies include immunising a mammal (e.g.mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or afragment thereof. Antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art, and screened,preferably using binding of antibody to antigen of interest. Forinstance, Western blotting techniques or immunoprecipitation may be used(Armitage et al., 1992, Nature 357: 80-82). Isolation of antibodiesand/or antibody-producing cells from an animal may be accompanied by astep of sacrificing the animal.

As an alternative or supplement to immunising a mammal with a peptide,an antibody specific for a protein may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, e.g.using lambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see WO 92/01047. The library may be naive, that is constructedfrom sequences obtained from an organism which has not been immunisedwith any of the proteins (or fragments), or may be one constructed usingsequences obtained from an organism which has been exposed to theantigen of interest.

Antibodies according to the present invention may be modified in anumber of ways. Indeed the term “antibody” should be construed ascovering any binding substance having a binding domain with the requiredspecificity. Thus the invention covers antibody fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimics that of an antibody enablingit to bind an antigen or epitope.

Example antibody fragments, capable of binding an antigen or otherbinding partner are the Fab fragment consisting of the VL, VH, C1 andCH1 domains; the Fd fragment consisting of the VH and CH1 domains; theFv fragment consisting of the VL and VH domains of a single arm of anantibody; the dAb fragment which consists of a VH domain; isolated CDRregions and F(ab′)2 fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

A hybridoma producing a monoclonal antibody according to the presentinvention may be subject to genetic mutation or other changes. It willfurther be understood by those skilled in the art that a monoclonalantibody can be subjected to the techniques of recombinant DNAtechnology to produce other antibodies or chimeric molecules whichretain the specificity of the original antibody. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe complementarity determining regions (CDRs), of an antibody to theconstant regions, or constant regions plus framework regions, of adifferent immunoglobulin. See, for instance, EP 0 184 187 A, GB 2 188638 A or EP 0 239 400 A. Cloning and expression of chimeric antibodiesare described in EP 0 120 694 A and EP 0 125 023 A.

Hybridomas capable of producing antibody with desired bindingcharacteristics are within the scope of the present invention, as arehost cells, eukaryotic or prokaryotic, containing nucleic acid encodingantibodies (including antibody fragments) and capable of theirexpression. The invention also provides methods of production of theantibodies including growing a cell capable of producing the antibodyunder conditions in which the antibody is produced, and preferablysecreted.

The reactivities of antibodies on a sample may be determined by anyappropriate means. Tagging with individual reporter molecules is onepossibility. The reporter molecules may directly or indirectly generatedetectable, and preferably measurable, signals. The linkage of reportermolecules may be directly or indirectly, covalently, e.g. via a peptidebond or non-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule. The mode of determining binding is not a feature of thepresent invention and those skilled in the art are able to choose asuitable mode according to their preference and general knowledge.

Other candidate inhibitor compounds may be based on modelling the3-dimensional structure of a polypeptide or peptide fragment and usingrational drug design to provide potential inhibitor compounds withparticular molecular shape, size and charge characteristics.

Mass Spectroscopy

An LC/MS/MS based strategy was used to discover new phosphorylationsites within tau protein isolated from AD brain. So called PHF-tau wasinitially extracted from a heat-stable preparation of human AD brainmaterial and subsequently further purified by ion exchangechromatography. Having been separated using SDS-PAGE, phospho-peptidemapping was then undertaken. Coomassie stained bands are excised,reduced, alkylated and enzymatically digested using a suite of proteasessuch as trypsin, chymotrypsin and endoproteinase Asp-N. Resultingpeptide mixtures are then analysed by LC/MS/MS using a Q-TOF microinstrument with peptide separation achieved using a 75 micron ID PepMapreversed phase column with peptides eluted using a gradient ofacetonitrile at a flowrate of 200 nl/min.

Database searching against bespoke index files is performed utilisingthe Mascot algorithm (Matrix Science). All MS/MS spectra relating tophosphopeptides are then subsequently visually verified to check theresult.

Tandem MS/MS of peptides may be used to provide sequence information byvirtue of the fragment ions produced. Fragmentation occurs generallyacross the peptide bond leading to a ladder of sequence ions that arediagnostic of the amino acid sequence. The difference betweenconsecutive ions in a series indicates the mass of the amino acid atthat position in the peptide. The most common ion types are b and yions. The C-terminal containing fragments are designated y-ions and theN-terminal containing fragments are designated b-ions (Roepstorff, P.,Fohlman, J. J. Biomed. Mass Spectrom. 1984, 11, 601). Peptides createdby trypsin proteolysis and ionised by electrospray generally form ionsthat are doubly charged. This stems from the presence of basic groupswithin the peptide, namely, the alpha amino group at the N-terminus andthe side chain of the C-terminal lysine or arginine. MS/MS spectra ofsuch peptides generally yield a prominent y-type ion series in the highmass end of the spectrum (Bonner, R., Shushan, B. Rapid Commun. MassSpectrom. 1995, 9, 1067-1076). Ideally, for de novo sequencing purposes,a complete set of complementary b and y ions will ensure a highconfidence level for the proposed peptide sequence. Moreover, iffragment ions representing the complete sequence of the peptide arepresent, the site of attachment of the phosphate group can be deducedfrom the position and pattern of these fragment ions. Therefore, it ispossible in most instances to discover the exact site of phosphorylationin each phosphopeptide. In some instances we have even found MS/MSspectra to be heterogeneous. Here two (or more) distinct phosphopeptidesare represented in the same spectrum. This is because eachphosphopeptide form has the same molecule weight and the same number ofphosphate groups, but these are attached to different amino acids withinthe peptide. Therefore, both forms give rise to precursor ions of thesame m/z ratio, which are then selected simultaneously by the massspectrometer during the MS/MS experiment. In such cases, we refer to thephosphopeptides concerned as “regiomers”

Multiplex Assays for Screening Compounds

In drug development it is desirable to develop rapid high throughputassays with simple read out to show whether a compound has an effect onthe proposed target. In the case of compounds inhibiting an enzymefunction, such as a kinase, it is possible to develop an artificialsubstrate for the target enzyme that is modified by the enzyme in a waythat the level of modification can be readily detected. In the presenceof an inhibitory compound, the substrate is not modified and this canalso be readily detected.

In the case of inhibitors of tau phosphorylation, it is necessary tomonitor the effect of inhibiting specific protein kinases on thephosphorylation status of a large number of sites. In one aspect, it ispossible to prepare artificial substrates corresponding to each of thephosphorylation sites on tau and assess each compound for their abilityto inhibit the phosphorylation of each site independent of the othersites. In such a system, each compound would be added to multiple wellseach well containing the proposed kinase target, one of thephosphorylation site-specific artificial substrates and a reportersystem to show phosphorylation, such as a monoclonal antibody that bindsspecifically to the substrate in either the phosphorylated orunphosphorylated form, and which antibody is labelled with a fluorescentmarker, an enzyme that converts a colour less substrate into a colouredproduct, or an enzyme that promotes the production of a luminescentsignal. In such an assay, it is desirable that the artificial substratefor the target is immobilised on a solid surface such that as part ofthe assay procedure any unreacted antibody is removed from the system bywashing before the result is read. Such assays may be run in microtitrewells of varying formats of typically 96, or more typically 384, or evenmore typically 1536 wells, or alternatively may be run on a microarraybased on a solid support such as glass.

Alternatively, the effect of different kinase inhibitors on the globalphosphorylation status of tau may be designed. In such an assay, fulllength recombinant tau protein carrying no phosphorylations, or one ormore desirable phosphorylations may be used as the substrate.Alternatively, a mixture of equal amounts of all of the artificialsubstrates representing single phosphorylation sites may be used. Eachscreening assay will determine the effect of compounds on the inhibitionof one, two or more protein kinases with known activity for thephosphorylation of tau. As with the more simple assays described abovesubstrate, target kinase and compound are added to a well of amicrotitre plate and incubated with appropriate buffers and otherconstituents that permit the phosphorylation of substrate in the absenceof an inhibitory compound. The phosphorylation status of the substratemay then be determined using a mixture of antibodies or other moleculeswith specificity for individual phosphorylation sites on tau, whereinsuch antibodies or other molecules are each labelled with a uniquereporter such as a fluorescent dye or compounds with unique spectralproperties in infra-red, visible or ultraviolet spectra. After removalof antibodies that remain unbound to the phosphorylated substrate(s),levels of each specific reporter are determined using an appropriatereading device, and the levels of phosphorylation at each specific sitein tau is revealed by comparison with a control where no kinaseinhibitor was added.

In a preferred embodiment of such a multiplex screening assay, thesubstrate is dephosphorylated recombinant tau protein and the kinase isselected from CK1, CK2, GSK-3a, GSK-3b, PKA, CDK5, ERK1/2, SAPK1g,SAPK2a, SAPK2b, SAPK3, SAPK4, stress activated protein kinase familykinases (SAPKs) such as p38MAPK and JNK, MARK family kinases such as110K, cdc2, cdk2, PKC, PKN, TTK, PKB, DYRK, PK, CaMKII, PKD, or amixture of one of more these kinases. Reporter systems are preferablylabelled antibodies, typically monoclonal antibodies, for example thosethat can be obtained from rabbits or mice using techniques well known inthe art. Labels are preferably fluorescent or colorimetric compoundsthat are covalently attached to antibodies, more preferably fluorescentor colorimetric nanoparticles and are most preferably nanoparticles withunique Raman spectra.

Development of Mimetic Substances

Once candidate substance have been found in the assays and screensaccording to the present invention, they may be used to design mimeticcompounds for development as drugs. The designing of mimetics to a knownpharmaceutically active compound is a known approach to the developmentof pharmaceuticals based on a “lead” compound. This might be desirablewhere the active compound is difficult or expensive to synthesise orwhere it is unsuitable for a particular method of administration, e.g.peptides are unsuitable active agents for oral compositions as they tendto be quickly degraded by proteases in the alimentary canal. Mimeticdesign, synthesis and testing is generally used to avoid randomlyscreening large number of molecules for a target property.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. Firstly, the particular partsof the compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,e.g. by substituting each residue in turn. These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its structure is modelledaccording to its physical properties, e.g. stereochemistry, bonding,size and/or charge, using data from a range of sources, eg spectroscopictechniques, X-ray diffraction data and NMR. Computational analysis,similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this in the design of themimetic.

A template molecule is then selected onto which chemical groups whichmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted on to it can conveniently be selected so thatthe mimetic is easy to synthesise, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimisation ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

Inhibitors

The term “inhibitor” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes theexpression or kinase activity of a kinase. Preferably, a kinaseinhibitor is a specific or near-specific inhibitor which inhibits theexpression or activity a desired kinase without affecting other kinases.

Kinase inhibitors include antibodies, dominant negative forms and smallmolecule inhibitors.

Small molecule inhibitors of Abl activity include phenylaminopyrimidinessuch as imatinib or imatinib mesylate (Glivec/Gleevec™,piperazinyl)methyl]-N-(4-methyl-3-[(4-(3-pyridinyl)-2-pyrimidinyl[amino]-phenyl]benzamidemethanesulfonate; Novartis); BMS-354825[n-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carbozamide];PD 173955 (Parke Davis); pyridopyrimidines such as PD166326 (ParkeDavis); ON 012380 (Onconova).

Small molecule inhibitors of Syk activity include picetannol(3,4,3′,5′-tetrahydroxy-trans-stilbene); 574711(3-(1-Methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide,Calbiochem); ER-27319; and BAY61-3606.

Small molecule inhibitors of Fyn activity include PP1(4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine).

Inhibitors of kinase expression include antisense RNA or siRNA asdescribed below, triple-helix nucleic acids or ribozymes.

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra; Lee et al., Nucl.Acids Res., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988);Dervan et al., Science, 251:1360 (1991).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Antisense

Expression of nucleic acid sequences that are complementary in sequenceto a coding sequence of a gene (‘antisense’ nucleic acids) can inhibitproduction of the protein product from the gene. It is not known exactlyhow this occurs, but it is thought that the antisense nucleic acidsequences hybridise to cellular mRNA, forming a double strandedmolecule. The cell does not translate the mRNA in this double-strandedform, so translation is inhibited. Antisense nucleic acids may haveother effects, including inhibition of transcription and splicinginhibition.

The term ‘antisense’ nucleic acid indicates a nucleic acid sequencewhich is sufficiently complementary to the RNA molecule for which theantisense nucleic acid is specific to cause molecular hybridisationbetween the antisense nucleic acid and the mRNA such that translation ofthe mRNA is inhibited. Such hybridisation must occur under in vivoconditions, that is, inside the cell.

Oligomers of about fifteen nucleotides or greater and molecules thathybridise to the AUG initiation codon are particularly efficient, sincethey are easy to synthesize and are likely to pose fewer problems thanlarger molecules when introducing them into cells.

RNA Interference

RNA interference (RNAi) is a process of sequence-specific,post-transcriptional gene silencing in animals and plants, initiated bydouble-stranded RNA (dsRNA) that is homologous in sequence to thesilenced gene. RNAi is mediated by short double-stranded RNA molecules(small interfering RNAs or siRNAs). siRNAs may be introduced into a cellas short RNA oligonucleotides of 10-15 bp, or as longer dsRNAs which aresubsequently cleaved to produce siRNAs. The RNA may be introduced intothe cell as RNA, or may be transcribed from a DNA or RNA vector.

Methods relating to the use of RNAi to silence genes in C. elegans,Drosophila, plants, and mammals are known in the art (Fire A, et al.,1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999);Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001);Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119 (2001); Tuschl,T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A. et al., Science 286,950-952 (1999); Hammond, S. M., et al., Nature 404, 293-296 (2000);Zamore, P. D., et al., Cell 101, 25-33 (2000); Bernstein, E., et al.,Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev. 15,188-200 (2001); WO0129058; WO9932619, and Elbashir S M, et al., 2001Nature 411:494-498).

In some embodiments, the siRNA has an overhang at one or both ends ofone or more deoxythymidine bases. The overhang is not to be interpretedas part of the siRNA sequence. Where present, it serves to increase thestability of the siRNA within cells by reducing its susceptibility todegradation by nucleases.

siRNA molecules may be synthesized using standard solid or solutionphase synthesis techniques which are known in the art. Linkages betweennucleotides may be phosphodiester bonds or alternatives, for example,linking groups of the formula P(O)S, (thioate); P(S)S, (dithioate);P(O)NR′2; P(O)R′; P(O)OR6; CO; or CONR′2 wherein R is H (or a salt) oralkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotidesthrough-O-or-S—.

Alternatively, siRNA molecules or longer dsRNA molecules may be maderecombinantly by transcription of a nucleic acid sequence, preferablycontained within a vector as described below.

Another alternative is the expression of a short hairpin RNA molecule(shRNA) in the cell. shRNAs are more stable than synthetic siRNAs. AshRNA consists of short inverted repeats separated by a small loopsequence. One inverted repeat is complimentary to the gene target. TheshRNA is then processed into an siRNA which degrades the target genemRNA and suppresses expression. shRNAs can produced within a cell bytransfecting the cell with a DNA construct encoding the shRNA sequenceunder control of a RNA polymerase III promoter, such as the human H1 or7SK promoter. Alternatively, the shRNA may be synthesised exogenouslyand introduced directly into the cell. Preferably, the shRNA sequence isbetween 40 and 100 bases in length, more preferably between 40 and 70bases in length. The stem of the hairpin is preferably between 19 and 30base pairs in length. The stem may contain G-U pairings to stabilise thehairpin structure.

Modified nucleotide bases can be used in addition to the naturallyoccurring bases, and may confer advantageous properties on siRNAmolecules containing them.

For example, modified bases may increase the stability of the siRNAmolecule, thereby reducing the amount required for silencing. Theprovision of modified bases may also provide siRNA molecules which aremore, or less, stable than unmodified siRNA.

The term ‘modified nucleotide base’ encompasses nucleotides with acovalently modified base and/or sugar. For example, modified nucleotidesinclude nucleotides having sugars which are covalently attached to lowmolecular weight organic groups other than a hydroxyl group at the 3′position and other than a phosphate group at the 5′ position. Thusmodified nucleotides may also include 2′ substituted sugars such as2′-O-methyl-; 2-O-alkyl; 2-O-allyl; 2′-S-alkyl; 2′-S-allyl; 2′-fluoro-;2′-halo or 2; azido-ribose, carbocyclic sugar analogues a-anomericsugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, and sedoheptulose.

Modified nucleotides are known in the art and include alkylated purinesand pyrimidines, acylated purines and pyrimidines, and otherheterocycles. These classes of pyrimidines and purines are known in theart and include pseudoisocytosine, N4,N4-ethanocytosine,8-hydroxy-N-6-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5 fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1-methyladenine,1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine,2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine,N6-methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxyamino methyl-2-thiouracil, -D-mannosylqueosine,5-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid methylester, psuedouracil, 2-thiocytosine, 5-methyl-2 thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acidmethylester, uracil 5-oxyacetic acid, queosine, 2-thiocytosine,5-propyluracil, 5-propylcytosine, 5-ethyluracil, 5-ethylcytosine,5-butyluracil, 5-pentyluracil, 5-pentylcytosine, and 2,6,diaminopurine,methylpsuedouracil, 1-methylguanine, 1-methylcytosine.

Pharmaceutical Compositions

Following identification of a substance which modulates or affectsphosphorylation or dephosphorylation of tau protein, the substance maybe investigated further. Furthermore, it may be manufactured and/or usedin preparation, i.e. manufacture or formulation, of a composition suchas a medicament, pharmaceutical composition or drug. These may beadministered to individuals.

Thus, the present invention extends in various aspects not only to asubstance identified using the screening assays and assay methodsdisclosed herein, but also a pharmaceutical composition, medicament,drug or other composition comprising such a substance, a methodcomprising administration of such a composition to a patient, e.g. totreat tauopathies, use of such a substance in manufacture of acomposition for administration for the treatment of tauopathies, and amethod of making a pharmaceutical composition comprising admixing such asubstance with a pharmaceutically acceptable excipient, vehicle orcarrier, and optionally other ingredients.

The substances identified as kinase inhibitors or phosphatase promotersin the assays and assay methods of the present invention, or compoundsor substances arising from further development or optimisation, may beformulated in pharmaceutical compositions. These compositions may beemployed for the treatment of tauopathies, that is conditions which arecharacterised by neurofibrillary tangles or aggregates of tau protein.Tauopathies are a recognised class of conditions known to those skilledin the art and include Alzheimer's disease (AD), frontotemproal dementiawith Parkinsonism linked to chromosome 17 (FTDP-17), progressivesupranuclear palsy (PSP), Pick's disease, corticobasal degeneration andmultiple system atrophy (MSA). The intracellular tau deposits areusually neuronal or glial and are filamentous and generally in ahyperphosphorylated state as compared to the level of phosphorylation intau from control human brain. In the case of AD, thishyperphosphorylated tau is often referred to as paired helical filamenttau (PHF) tau because it is derived from the PHF.

These compositions may comprise, in addition to one of the abovesubstances, a pharmaceutically acceptable excipient, carrier, buffer,stabiliser or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the active ingredient. The precise nature of the carrier orother material may depend on the route of administration, e.g. oral,intravenous, cutaneous or subcutaneous, nasal, intramuscular,intraperitoneal routes.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Whether it is a polypeptide, antibody, peptide, nucleic acid molecule,small molecule or other pharmaceutically useful compound according tothe present invention that is to be given to an individual,administration is preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to pfactitioners.Examples of the techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 20th Edition, 2000, pub.Lippincott, Williams & Wilkins. A composition may be administered aloneor in combination with other treatments, either simultaneously orsequentially, dependent upon the condition to be treated.

Experimental

Purification of PHF-Tau from Alzheimer Brain

Paired helical filament (PHF) tau was purified from Alzheimer brain asdescribed in Hanger et al, 1998. Briefly, brain tissue was homogenisedand insoluble PHF-tau was recovered by differential centrifugation.Following solubilisation in guanidine and dialysis against a re-naturingbuffer, PHF-tau was purified by anion-exchange and reversed-phasechromatography.

Preparation of Mutant Forms of Human Tau

To generate the five tau constructs each with a single tyrosine replacedby phenylalanine, a QuikChange XL site-directed mutagenesis kit(Stratagene, Amsterdam, The Netherlands) was used. Primers(custom-synthesised by Sigma-Genosis) were as follows: to convert Tyr-18to Phe (giving tau construct Y18F), forward primer 5′-CAC GCT GGG ACGTTC GGG TTG GGG GAC-3′ (Primer A), and reverse primer 5′-GTC CCC CAA CCCGAA CGT CCC AGC GTG-3′; to convert Tyr-29 to Phe (giving Y29F), forwardprimer 5′-GAT CAG GGG GGC TTC ACC ATG CAC CAA G-3′ (Primer B), andreverse primer 5′-C TTG GTG CAT GGT GAA GCC CCC CTG ATC-3′; to convertTyr-197 to Phe (giving Y197F), 5′-GAT CGC AGC GGC TTC AGC AGC CCC GG-3′(Primer C), and reverse primer 5′-CC GGG GCT GCT GAA GCC GCT GCG ATC-3′;to convert Tyr-310 to Phe (giving Y310F), forward primer 5′-GGC AGT GTGCAA ATA GTC TTC AAA CCA GTT GAC CTG AG-3′ (Primer D), and reverse primer5′-CT CAG GTC AAC TGG TTT GAA GAC TAT TTG CAC ACT GCC-3′; and to convertTyr-394 to Phe (giving Y394F), forward primer 5′-GCG GAG ATC GTG TTC AAGTCG CCA GTG G-3′ (Primer E), and reverse primer 5′-C CAC TGG CGA CTT GAACAC GAT CTC CGC-3′. The sequence of the full insert was determined foreach construct (Lark Technologies).

To change all five tyrosines to phenylalanines a QuikChange® multisite-directed mutagenesis kit (Stratagene) was used, with the fiveprimers A to E (above). Colonies were sequenced, and as well asidentifying constructs where all five tyrosines had been replaced byphenylalanine (TauYallF), constructs with a single tyrosine remainingwere found that contained four phenylalanines and just Tyr-18, Tyr-29 orTyr-197. Mutants containing just Tyr-310 or just Tyr-394 were generatedfrom the all-Phe construct by single site-directed mutagenesis as aboveusing the following primers: for Tyr-310 only, forward primer 5′-GGC AGTGTG CAA ATA GTC TAC AAA CCA GTT GAC CTG AG-3′, and reverse primer 5′-CTCAG GTC AAC TGG TTT GTA GAC TAT TTG CAC ACT GCC-3′; and for Tyr-394only, forward primer 5′-GCG GAG ATC GTG TAC AAG TCG CCA GTG G-3′, andreverse primer 5′-C CAC TGG CGA CTT GTA CAC GAT CTC CGC-3′. The fiveconstructs with one remaining tyrosine were termed Y18 only, Y29 only,etc, and their tau coding sequences verified (Lark Technologies).

Preparation and Purification of Recombinant Human Tau

A plasmid expressing the largest tau isoform (2N4R) was used to prepareand purify recombinant human tau as described previously (Mulot et al.,1994). Briefly, a bacterial cell lysate expressing 2N4R tau was heatedand centrifuged to remove heat-labile proteins. The supernatant wasfractionated with ammonium sulphate and precipitated material wassolubilised and dialysed into buffer prior to cation-exchangechromatography. Proteins were eluted with NaCl and fractions containingtau were pooled and dialysed against Mes buffer pH 6.25, 5 mM DTT, andstored frozen.

In Vitro Phosphorylation of Recombinant Tau by Rat Brain Lysate

A rat brain extract containing active protein kinases was prepared byhomogenising a rat brain in ice-cold buffer (2 ml buffer per g brain)containing 25 mM Tris-HCl pH7.5, 5 mM EGTA, 2 mM dithiothreitol (DTT), 2μM okadaic acid, 1 mM sodium orthovanadate and protease inhibitors. Thehomogenate was centrifuged at 100,000 g for 1 hr, and incubated on icefor 30 min with 2 mM ATP and 10 μM okadaic acid. This extract (rat brainsupernatant, RBS) contained 7 mg/ml protein (Bradford).

Recombinant 2N4R tau protein (100 μg/ml) was phosphorylated byincubating with RBS (1.8 mg/ml protein) in 50 mM Tris-HCl buffer pH 7.5with 5 mM MgCl₂, 3 mM ATP, 5 mM EGTA, 1 mM sodium orthovanadate, 10 μMokadaic acid, 1 mM DTT and protease inhibitors at 37° C. for 24 hr. Thereaction mixture was then heated at 100° C. for 5 min, incubated on icefor 10 min, and centrifuged for 10 min at 16000 g. The supernatantcontaining the tau protein was aspirated off and analysed by Westernblotting and mass spectrometry.

In Vitro Phosphorylation of Recombinant Tau by Tyrosine Protein Kinases

Recombinant human tau (1 μg) was incubated with 50 ng of either Abl orSyk (Upstate) in 30 μl of kinase buffer (HEPES 50 mM pH 7.4, 10 mM MnCl₂in the presence of 1 mM ATP) for 30 minutes at 30° C. 30 μl of SDS-PAGEsample buffer was added to stop the reaction.

In Vitro Phosphorylation of Recombinant Tau by Lck and SubsequentBinding of SH2 Domain of Fyn

Recombinant 2N4R tau protein (440 μg/ml) was incubated at 30° withpurified recombinant Lck (20-100 μg/ml), 40 mM β-glycerophosphate bufferpH 7.5, 3 mM ATP, 25 mM MgCl₂, 5 mM MnCl₂, 1 mM DTT, 100 μM EDTA, 1 mMsodium orthovanadate and protease inhibitors. After 6 hr the tubes wereheated at 100° for 5 min, cooled for 10 min on ice, and centrifuged for10 min at 16000 g. The supernatants were checked for tyrosinephosphorylation by Western blotting, using 4G10 anti-phosphotyrosineantibody (Upstate, Inc) and anti-tau antibody (Dako).

Interaction of tau with the SH2 domain of Fyn was investigated byincubating tyrosine-phosphorylated or non-phosphorylated tau (5 μg/ml)with glutathione-Sepharose® beads containing 2-5 μg of GST-Fyn-SH2fusion protein or GST as control. After mixing for 60 min at 4° thebeads were washed x3 and analysed by Western blotting for tau and forphosphotyrosine as above.

In-Gel Proteolytic Digestion of Tau

PHF-tau or in vitro phosphorylated tau proteins were separated on 10%(wt/vol) polyacrylamide gels and stained with colloidal Coomassie BlueG. Protein bands corresponding to tau were excised, carbamidomethylated,and digested with proteolytic enzymes (trypsin or Asp-N). Peptides wereextracted from gel pieces by a series of acetonitrile and aqueouswashes, dried and resuspended in 50 mM ammonium bicarbonate.

Amyloid Beta Treatment of Neurons and Isolation of Lipid Rafts

Rat and human primary cortical neuronal cultures were treated withfibrillar Aβ₂₅₋₃₅ or Aβ₁₋₄₂ for 1-30 min. Lipid rafts were prepared fromcontrol untreated and Aβ-treated neuronal cultures by scraping the cellsfrom one 80 cm² flask into 2 ml 1% Triton X-100 in 25 mM Mes, pH 6.5containing protease inhibitors. Cells were disrupted by Douncehomgenization. The homogenate was mixed with 2 ml of 90% sucrose (w/v)in 25 mM Mes, 150 mM NaCl pH 6.5 and placed in a 12 ml centrifuge tube.A 5-35% step sucrose gradient was formed by overlaying the homogenatemix with 4 ml of a 35% (w/v) sucrose solution followed by 4 ml of a 5%(w/v) sucrose solution. This was then centrifuged at 39,000 rpm for 18hr in a Beckman SW41 rotor. 1 ml fractions were collected from the topof each gradient. The lipid raft fraction partitioned at the interfacebetween the 5% (w/v) sucrose layer and the 35% sucrose (w/v) layer,fractions 4 and 5. Lipid raft fractions were concentrated by mixing theraft fractions (4 and 5) with 10 ml dd H₂O and centrifuging at 39,000rpm for 2 h in a Beckman SW41 rotor. The supernatant was aspirated andthe remaining pellet was resuspended in 100 μl of 2× sample buffer.Western blots of lipid raft proteins were probed for antibodies toflotillin and protein loading corrected by scanning densitometry. Tauwas detected in lipid rafts by probing western blots of lipid raftproteins using a polyclonal anti-tau antibody (DAKO).

Phosphorylation of Tyrosine Residues in Tau in Cultured Cells Treatedwith Pervanadate

In a first set of experiments, COS-7 cells were transiently transfectedwith V5 tagged human tau longest isoform or with V5 tagged mutants oftau where one tyrosine has been replaced by one phenylalanine (namedY18F, Y29F, Y197F, Y310F and Y394F) constructs. In a second set ofexperiments, COS-7 cells were transiently transfected with the V5 taggedtau (441) construct or with V5 tagged mutants of tau where only onetyrosine is remaining, the four other tyrosines being replaced byphenyalanine (named Y18-only, Y29-only, Y197-only, Y310-only andY394-only according to the remaining tyrosine). In order to increase tautyrosine phosphorylation, cells were treated with the tyrosinephosphatase inhibitor pervanadate for 20 minutes. Cells were harvestedin NETF buffer (100 mM NaCl, 2 mM EGTA, 50 mM Tris-Cl pH 7.4 and 50 mMNaF) containing 1% NP-40, 2 mM orthovanadate and protease inhibitors.Samples were precleared with 40 μl of protein G-Sepharose beads, andimmunoprecipitations were carried out with monoclonal anti-V5 antibodiespreadsorbed on protein G-Sepharose beads. Cells were harvested in NETFlysis buffer containing 1% NP-40 and tau was immunoprecipitated using ananti-V5 antibody. Resulting immunoprecipitates were separated induplicate by SDS-PAGE and transferred to nitrocellulose. Immunoblotswere performed on duplicate membranes using 4G10 phosphotyrosineantibody or TP70 antibody (total tau antibody). Bound antibodies werevisualized by enhanced chemiluminescence detection. Quantification wasachieved by scanning the autoradiograms with GS710 Calibrated ImagingDensitometer (Bio-Rad) and measurement of relative optical density withQuantity One 4.0.3 software (Bio-Rad).

Phosphorylation of Tyrosine Residues in Tau in Cultured Cells byCo-Expression of Tyrosine Kinases, Fyn, Src, Abl, Syk

Fyn cDNA was a gift from D. Markby (Sugen, San Francisco), Src cDNA wasfrom upstate (Src cDNA allelic pack), Abl and AblΔXB cDNA (aconstitutively active form of Abl, with deletion of most of the SH3domain) were from Richard A. Van Etten (Molecular Oncology ResearchInstitute, Boston), Syk cDNA was a gift from H. Band (Brigham andWomen's Hospital, Boston). CHO cells were used for the co-transfectionexperiments. CHO cells were transiently transfected with the V5 taggedhuman tau longest isoform and with V5 tagged mutants of tau where onetyrosine has been replaced by one phenylalalanine (named Y18F, Y29F,Y197F, Y310F and Y394F) constructs. In every experiment, cells wereco-transfected with the empty vector or with the protein tyrosine kinaseexpression vector (Fyn, Src, Abl or AblΔXB). Harvesting of cells,immunoprecipitation and Western analysis were performed as described inthe section “Phosphorylation of tyrosine residues in tau in culturedcells treated with pervanadate”.

Results New Sites of Tyrosine Phosphorylation Found in PHF-Tau

Current literature reports 25 known phosphorylation sites (all areserine or threonine) identified by direct means in PHF-tau (Hanger etal, 1998) (Morishima-Kawashima et al., 1995). There are a further 2-3sites that have been identified by antibody reactivity only. On thebasis of antibody labelling, it has been reported that tyrosine 18 isphosphorylated in a proportion of PHF-tau in AD brain. We have found anadditional 12 phosphorylation sites in PHF-tau, one of which is atyrosine residue (tyr394), bringing the total number of sites to 37. Wehave also found that tyr394 is phosphorylated in tau isolated from humanfoetal brain.

New Sites of Tyrosine Phosphorylation on Recombinant Tau Generated byRat Brain Lysate

Mass spectrometry of proteolytic digests of tau that had beenphosphorylated with rat brain supernatant demonstrated phosphorylationon tyrosines 310 and 394, in addition to many serines and threonines.

Aβ Treatment of Neurons and Lipid Raft Composition

Aβ₂₅₋₃₅ and Aβ₁₋₄₂ treatment of primary rat neuronal cultures resultedin a rapid increase in the tyrosine phosphorylation of neuronal proteincomponents of lipid rafts. No increase in the phosphoserine orphosphothreonine content of lipid raft proteins was observed afterAβ-treatment. The increase in tyrosine phosphorylation was concomitantwith an increased partitioning of Fyn, tau, and tubulin into lipidrafts. Focal adhesion kinase (FAK) levels transiently increased in lipidrafts in response to Aβ while levels of the classic lipid raft proteinflotillin remained unchanged. Inhibition of tyrosine phosphorylationwith the tyrosine kinase inhibitor PP2 abrogated the Aβ-induced increasein tyrosine phosphorylation of lipid raft proteins and partitioning oftau into lipid rafts.

Phosphorylation of Tyrosine Residues in Tau in Cultured Cells Treatedwith Pervanadate

The first set of five mutants where one tyrosine residue was exchangedwith phenylalanine (Y18F, Y29F, Y197F, Y310F and Y394F) were transfectedinto COS-7 cells and cells were treated for 20 minutes with pervanadate.Western analysis performed on immunoprecipitated tau, using 4G10antiphosphotyrosine antibody shows that the Y394F mutant construct isthe only single tyrosine mutation that results in a significant effect,reducing tyrosine phosphorylation to approximately 10% of the wild-typecontrol. Tyrosine phosphorylation of the Y18F, Y29F, Y310F constructswere not significantly different from the wild-type control. Concerningthe Y197F mutant construct, it should be pointed out that a decrease intyrosine phosphorylation was observed in two of the five experimentsthat were done with this construct. To confirm these results, wetransfected into COS-7 cells the second set of mutants in which only onetyrosine residue remains as the sole tyrosine with the other fourreplaced by phenylalanine (Y18-only, Y29-only, Y197-only, Y310-only andY394-only). Analysis using phosphotyrosine antibodies showed that notyrosine phosphorylation could be elicited by pervanadate in Y18-only,Y29-only, Y310-only mutant constructs, whereas pervanadate induces anincrease in tyrosine phosphorylation of the Y394-only similar to the oneobserved in wild-type tau. In two of the four experiments made with theY197-only mutant construct, a faint but clear-cut phosphotyrosineimmunoreactivity was detectable. Taken together, these results suggestthat the majority of tyrosine phosphorylation of tau inpervanadate-treated COS-7 cells occurs on tyrosine 394.

Phosphorylation of Tyrosine Residues in Tau in Cultured Cells OverExpressing Fyn

Wild type tau and the first set of five mutants where one tyrosineresidue was exchanged with phenylalanine (Y18F, Y29F, Y197F, Y310F andY394F) were co-transfected with the empty vector or with aFyn-expression vector into CHO cells. Western analysis performed onimmunoprecipitated tau, using 4G10 phosphotyrosine antibody, shows thatthe Y18F and Y310F mutant constructs are the two single tyrosinemutations that results in a significant effect, each mutation reducingtyrosine phosphorylation to approximately 50% of the wild-type control.Taken together, the results suggest that tyrosine 18 and 310 are themain sites phosphorylated by Fyn.

Phosphorylation of Tyrosine Residues in Tau in Cultured CellsOver-Expressing Abl

Wild type tau and the first set of five mutants where one tyrosineresidue was exchanged with phenylalanine (Y18F, Y29F, Y197F, Y310F andY394F) were co-transfected with the empty vector or with AblΔXBexpression vector into CHO cells. Western analysis performed onimmunoprecipitated tau, using 4G10 phosphotyrosine antibodies, showsthat the Y394F is the tyrosine mutation with the strongest effectreducing tyrosine phosphorylation to approximately 25% of the wild-typecontrol. Y197F and Y310F mutant constructs also have a significanteffect reducing tyrosine phosphorylation to approximately 70% of thewild-type control each. In contrast, Y18F and Y29F mutant constructswere not different from the wild type control. Taken together, theresults suggest that Abl primarily phosphorylates tau on tyrosine 394and that tyrosines 197 and 310 are also phosphorylated by this kinase.In contrast, Abl does not phosphorylate tyrosines 18 and 29.

Phosphorylation of Tyrosine Residues in Tau in Cultured NeuronsOver-Expressing Syk

Wild type tau and the first set of five mutants where one tyrosineresidue was exchanged with phenylalanine (Y18F, Y29F, Y197F, Y310F andY394F) were co-transfected with the empty vector or with a Sykexpression vector into CHO cells. Western analysis performed onimmunoprecipitated tau, using 4G10 phosphotyrosine antibodies, showsthat single mutants of tau (i.e. with one tyrosine mutated tophenylalanine, the other four tyrosines still being present) showed nosignificant decreases in tau tyrosine phosphorylation. Mutants with onlyY18, Y29, Y197 or Y394 could each be phosphorylated to 20-25% of thelevel found with wild-type, indicating that Syk can phosphorylate tau ateach of these sites.

SH2 Domain of Fyn Binding to Tyrosine-Phosphorylated Tau

Co-sedimentation experiments using GST-SH2 proteins bound to glutathionebeads demonstrated that tyrosine phosphorylated tau, but not controlnon-phosphorylated tau, could bind to the SH2 domain of Fyn (isoform B).

Use of STI 571 to Determine Whether Phosphorylation of Tau in Cells isCatalysed by an Abl-Like Kinase

The chemical compound STI 571, also known as Imatinib mesylate,Gleevec(R), Glivec, formerly CGP 57148B, chemical name4-[(4-Methyl-1-piperazinyl)methyl)-N-[4-methyl-3-[([4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamidemethanesulphonate, is a known inhibitor of tyrosine protein kinases andan effective antileukaemic agent. It is selective for Abl but alsoinhibits a small number of other tyrosine protein kinases including theplatelet-derived growth factor receptor and c-Kit.

An experiment to confirm whether an Abl-like kinase phosphorylates tauin cells could be carried out as follows. COS-7, CHO or SH-SY5Y cellsare transfected with a suitable tau construct, e.g. a plasmid containingTau2N4R-V5-His (wild-type) and after 48 hours treated with STI-571followed 1 hour later with 100 micromolar pervanadate or control.

After a further one hour cells are harvested and cell lysates areimmunoprecipitated with anti-V5 antibody and analysed by Westernblotting with the antiphosphotyrosine antibody 4G10. It would beexpected that, as already shown with the compound PP2, STI 571 willinhibit the tyrosine-phosphorylation of tau.

Hypothesis

Our hypothesis is that Aβ is neurotoxic to neurons by a mechanism thatobligatorily requires the involvement of tau and certain proteintyrosine kinases; likely candidate tyrosine kinases include Fyn and Ablbut others may be required. We envisage a mechanism in which exposure ofneurons to Aβ induces activation of one or more of these tyrosinekinases, which then phosphorylate tau and this generates binding sitesfor other cell signalling proteins, including for example an SH2 bindingsite for Fyn. Tyrosine phosphorylated tau then binds to lipid raftcomponents of cell membranes in amounts that are pathological and thistriggers unknown but detrimental cell signalling processes that resultin neurodegeneration and cell death.

REFERENCES

The references cited herein are all expressly incorporated by reference.

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1.-51. (canceled)
 52. An in vitro method of screening for substanceswhich are candidate therapeutic agents for the treatment of tauopathies,said substance being effective to inhibit the phosphorylation of a tauprotein by a tyrosine kinase, wherein the tau protein comprises at leastone phosphorylation site, the method comprising: (a) contacting at leastone said substance, the tau protein and tyrosine kinase under conditionsin which the tyrosine kinase is capable of phosphorylating the site(s)of the tau protein in the absence of the substance; (b) detectingwhether, and optionally the extent to which, the substance inhibits thephosphorylation of the tau protein at one or more sites of the tauprotein by the tyrosine kinase; and, (c) selecting the substance whichinhibits phosphorylation of the tau protein at one or more of the sites;wherein the tyrosine kinase is selected from the group consisting of Fynor Syk.
 53. The method of claim 52, wherein the tau protein is pairedhelical filament tau.
 54. The method of 52, wherein the tau protein is afragment or derivative of a tau protein having the amino acid sequenceset out in FIG.
 1. 55. The method of claim 52, wherein the tau proteinhas greater than 80% sequence identity with a tau protein having theamino acid sequence set out in FIG.
 1. 56. The method of claim 52,wherein the tyrosine kinase phosphorylates tau protein at one or moresites selected from the group consisting of Y18, Y29, Y197, Y310 andY394 of tau protein.
 57. The method of claim 52, wherein the tyrosinekinase is Fyn.
 58. The method of claim 57, wherein Fyn phosphorylatestau protein at one or more sites selected from the group consisting ofY18 and Y310 of tau protein.
 59. The method of claim 52, wherein thetyrosine kinase is Syk.
 60. The method of claim 59, wherein Sykphosphorylates tau protein at Y18, Y29, Y197 and Y394 of tau protein.61. The method of claim 52, wherein the method comprises detecting instep (b) whether, and optionally the extent to which, the substanceundergoing screening inhibits the phosphorylation of a substrate by thetyrosine kinase.
 62. The method of claim 61, wherein the substrate ofthe tyrosine kinase is not a tau protein or a fragment thereof.
 63. Themethod of claim 52 wherein the method comprises determining in step (b)whether, and optionally the extent to which, the substance undergoingscreening inhibits the phosphorylation of a substrate by the caseinkinase
 1. 64. The method of claim 63, wherein the substrate of thetyrosine kinase is not a tau protein or a fragment thereof.
 65. Themethod of claim 63, wherein the method further comprises confirmingwhether a substance selected in an initial screen has the property ofinhibiting the phosphorylation of the tau protein under conditions inwhich the tyrosine kinase is capable of phosphorylating the site(s) ofthe tau protein in the absence of the substance.
 66. The method of claim52 wherein the step of determining the presence, absence or extent ofphosphorylation at one or more sites of the tau protein employs massspectroscopy or a site specific recognition agent which is capable ofdistinguishing between a phosphorylated and a non-phosphorylated site.67. The method of claim 66, wherein the site specific recognition agentis a monoclonal antibody.
 68. The method of claim 52, wherein thescreening is carried out in a multiplex assay employing a solid phase onwhich a plurality of substrates are immobilised.
 69. The method of claim68, wherein the substrates correspond to phosphorylation sites of tauprotein.
 70. The method of claim 69, wherein the phosphorylation sitesare one or more of the sites selected from the group consisting of Y18,Y29, Y197, Y310 and Y394 of tau protein.
 71. The method of claim 52, themethod comprising having identified a substance as an inhibitor of saidtyrosine kinase, the further step of optimizing the structure of thesubstance.
 72. A method which comprises having identified a substance bythe method of claim 52, the further step of manufacturing the substanceand/or formulating it in pharmaceutical composition.